We are studying protein secretion in E.coli. We propose to develop a system in which plasmid-cloned genes for secreted protein can be expressed under control of strong, regulated promotors of phage lambda. We have made such a plasmid containing lamB (the gene for lambda phage receptor, an outer membrane protein) and amp r (the gene for beta-lactamase, a periplasmic penicillinase). We have used this plasmid to show that these two proteins are made in vitro as precursor proteins with NH2-terminal hydrophobic peptides which are not present in the mature protein. We propose to use this plasmid in E.coli mini cells, aberrant E.coli cells which contain no chromosomal DNA but only plasmid DNA, to follow the sequence of events involved in the export of proteins across the cytoplasmic membrane. We will also use a plasmid system to generate in vitro mutations in the "signal sequence" of the two genes then use the mini cell system to study the effects of these alterations on the ability of the cell to secrete the proteins. We will also use the multicopy plasmid system to look for "dominant" mutations affecting the cells secretory machinery. We expect that these experiments will help us understand: (1) how secreted proteins are distinguished from non-secreted proteins, (2) the steps involved in secretion and (3) the components of the cell which carry out secretion.